Why You Might Want Excitation Intensity Control

 

Why would you want to vary the intensity of the excitation light source? In general you want as much fluorescence as you can get! Many naturally fluorescent subjects do not glow very brightly, and the fluorescent proteins and stains used in the life sciences can range from dim to bright but tend to be localized in small areas. The more fluoresced photons you can get out the better, and at times we are even asked how to get more intensity out of the system (for example, by using the Dual Light Head Hanger Kit).

But there are some instances in which there can be too much of a good thing, and for these excitation intensity control is a good feature:

• If your fluorophore is sensitive to photobleaching you want to use the minimum excitation needed to see the subject: any more just hastens the degradation;
• We have heard of isolated cases in which anesthetized fruit flies (Drosophila) did not ‘wake up’ when being sorted with the NIGHTSEA system. This can only be attributed to excessive heating of the subject due to absorption of the strong excitation light. This can be remedied by reducing the time that the flies are under the light, or by using the intensity control to reduce the excitation to the minimum level needed to do the sorting.
• Fluorescent indicators used in industry can be extremely bright, to the point that they are easier to observe if the intensity is reduced;
• If you want to balance fluorescence and reflected white light in an image to provide context you may need better control of the excitation intensity;
• There are situations in which a reduction in excitation intensity can result in an improvement in viewing contrast.

The last case is interesting. We encountered this while working with a customer interested in finding defects (gel) in nylon granules (read our article for more information on this application). The entire granule fluoresces and the small defect shows up as a brighter spot. The customer found that decreasing the excitation intensity produced an increase in viewing contrast. This is nicely illustrated in the two images below.

(Click image for larger view)

Reducing the excitation intensity reduces the resulting fluorescence over the full image area, but the fluorescence intensity of the background appears to decrease faster than that from the areas of interest. There is a ‘sweet spot’ that the operator could set for optimum viewing contrast. This certainly relates to non-linear responses of human visual perception to changes in intensity, and differences in those responses in areas of low and high intensity.